Complete mitochondrial genomes of two moths in the tribe Trichaeini (Lepidoptera: Crambidae) and their phylogenetic implications

Abstract The complete mitochondrial genomes of two Prophantis species in the tribe Trichaeini (Lepidoptera: Crambidae) were sequenced using high‐throughput sequencing technology. They were assembled and annotated: The complete mitogenomes of P. octoguttalis and P. adusta were 15,197 and 15,714 bp, respectively, and contain 13 protein‐coding genes (PCGs), 22 transfer RNA genes, two ribosomal RNA genes, and an A + T‐rich region. Their arrangement was consistent with the first sequenced mitogenome of Bombyx mori (Bombycidae) in Lepidoptera, which had the trnM–trnI–trnQ rearrangement. The nucleotide composition was obviously AT‐biased, and all PCGs, except for the cox1 gene (CGA), used ATN as the start codon. Except for trnS1, which lacked the DHU stem, all tRNA genes could fold into the clover‐leaf structure. The features of these two mitogenomes were highly consistent with those of other species of Spilomelinae in previous studies. Phylogenetic trees of Crambidae were reconstructed based on mitogenomic data using maximum likelihood and Bayesian inference analysis methods. Results showed that Trichaeini in this study robustly constitute a monophyletic group in Spilomelinae, with the relationships (Trichaeini + Nomophilini) + ((Spilomelini + (Hymeniini + Agroterini)) + Margaroniini). However, the affinities of the six subfamilies Acentropinae, Crambinae, Glaphyriinae, Odontiinae, Schoenobiinae, and Scopariinae within the “non‐PS Clade” in Crambidae remained doubtful with unstable topologies or low supports.


| INTRODUC TI ON
The two Prophantis species in the tribe Trichaeini in this study belong to Pyraloidea in Lepidoptera (Figure 1). The Pyraloidea, with more than 16,000 described species worldwide, is one of the largest groups in Lepidoptera, and it is composed of two families: Pyralidae and Crambidae, with Crambidae accounting for 60% of the species diversity (Munroe & Solis, 1999;Nuss et al., 2023). Regier et al. (2012) present a most detailed molecular estimate of relationships to date across the subfamilies of Pyraloidea based on five nuclear genes, in which the Crambidae was divided into three major lineages based on phylogenetic relationships: the "PS clade" (Pyraustinae, Spilomelinae, and Wurthiinae), the "OG clade" (Evergestinae, Glaphyriinae, Noordinae, and Odontiinae), and the "CAMMSS clade" (Acentropinae, Crambinae, Musotiminae, Midilinae, Scopariinae, and Schoenobiinae), forming a system of PS clade + (OG clade + CAMMSS clade). However, there is no stable and convincing phylogenetic relationship within "non-PS Clade" in the phylogenetic tree topology of the Pyraloidea based on nuclear or mitogenomic data in previous studies (Léger et al., 2021;Liu et al., 2021;Qi et al., 2021;Regier et al., 2012;Zhang et al., 2020). More molecular data, such as the mitogenomes, are in demand to reveal the phylogenetic relationships of the subfamilies in Crambidae.
Spilomelinae is the most species-rich subfamily in Crambidae, with 4135 described species in 344 genera (Nuss et al., 2023). species (Nuss et al., 2023). This tribe includes the genus Prophantis Warren, 1896, which consists of eight species that have all been poorly studied besides their original descriptions (Warren, 1896).
adusta Inoue, 1986 have been recorded from China. P. octoguttalis, the type species of the genus, is widespread, and is mainly distributed in southern China, Australia, India, and the Afrotropical region (Ratnasingham & Hebert, 2007;Wang, 1980). Its larvae feed on Coffea arabica (Linnaeus, 1757), and a single larva can harm several berries in succession, which can seriously impact coffee production (Wang, 1980). The adults of P. adusta are very similar in appearance to those of P. octoguttalis (Figure 1), which makes species identification in these moths very challenging.
The mitochondrial genome (mtDNA) is a closed-loop DNA double-helix molecule that varies significantly in length among taxa.
The mtDNA of lepidopteran insects is generally 15-16 kb in size and consists of 37 genes, including 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and a control region of variable length also known as A + T-rich region and D-loop region (Boore, 1999). Because of its conserved genetic components, compact arrangement, fast evolutionary rate, and maternal inheritance, it contains relevant genetic and developmental information that can be used in phylogenetic studies for different research purposes (Cameron, 2014;Wesley et al., 1979). The mtDNA has been widely used in molecular phylogeny, phylogeography, and genetic differentiation (Heise et al., 1995;Suzuki et al., 2013;Wang et al., 2019).
To date, only 23 mitogenomes of Spilomelinae have been published in GenBank, and no mitogenomes of Trichaeini have been reported. In this study, we sequenced the mitogenomes of P. octoguttalis and P. adusta of the Trichaeini for the first time, and performed preliminary bioinformatics analysis, including the gene size and arrangement, base composition, codon usage, and tRNA secondary structure, which can help us to understand the features of mitogenomes of Trichaeini and Spilomelinae. To understand the phylogenetic relationships of Spilomelinae and the position of Trichaeini, we reconstructed the phylogenetic tree based on the mitogenomes data of these two species with other available mitogenomes of Crambidae from GenBank by using maximum likelihood (ML) and Bayesian inference (BI) methods. It will provide new perspectives and genomic data for the phylogenetic research in Trichaeini and Spilomelinae.

| Specimen collection and DNA sequencing
The specimen of P. octoguttalis investigated was collected from

| Sequence assembly, annotation, and analysis
The high-quality data (clean data) of the samples, which were trimmed by BGI Genomics, were saved as fastq. format and imported into analyzed on Geneious Prime v2022.1.1. The published COI sequences of each species (MH418217 and KY370920) were downloaded from GenBank as reference sequences (Lopez-Vaamonde et al., 2019;Segar et al., 2017), and sequence extension was performed using the "Map to reference" function until repetitive base alignments appeared, indicating that the mitochondrial genome was assembled into a loop.
MAFFT (Multiple Alignment using Fast Fourier Transform) alignment was used to align the reference sequence with the F I G U R E 1 Visualization of the mitochondrial genomes of Prophantis octoguttalis and P. adusta. sample sequence, and PCGs were determined based on the similarity between genes. With the help of EditSeq v7.1.0, PCGs were translated into amino acids to further verify the correctness of the start codon, stop codon, and amino acid sequence, to ensure the accuracy of PCGs. The location and secondary structure of tRNA genes were predicted using the MITOS Web Server (Donath et al., 2019), and the chart of secondary structure was mapped using Adobe Illustrator v26.0. Ribosomal RNA genes are relatively conserved and can be determined by the position between the two genes (Boore, 2006). The A + T-rich region was generally located behind the rrnL gene. Mitogenome maps were generated using Proksee (https://proks ee.ca/). Sequence length, base composition, gene spacing, and overlap were viewed directly using Geneious Prime v2022.1.1. The base skew was calculated using the formula: AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) (Perna & Kocher, 1995). Relative synonymous codon usage (RSCU) was analyzed using MEGA v10.2.5.

| Phylogenetic analysis
A total of 55 mitogenome sequences (two from this study, 53 retrieved from GenBank) were used to construct the phylogenetic tree. The ingroups included five species of Acentropinae, five species of Crambinae, one species of Glaphyriinae, three species of Odontiinae, eight species of Pyraustinae, one species of Schoenobiinae, one species of Scopariinae, and 25 species of Spilomelinae. The four species (Lista haraldusalis, Galleria mellonella, Dioryctria yiai, and Pyralis farinalis) of Pyralidae, Bombyx mori of Bombycidae, and Helicoverpa armigera of Noctuidae were selected as outgroups (Table 1).
We used two datasets: (1) PCG123: all three codon positions of 13 PCGs; (2) PCG123RT: all three codon positions of 13 PCGs, two rRNA genes and 22 tRNA genes. ML and BI were used to construct phylogenetic trees.
ModelFinder (Kalyaanamoorthy et al., 2017) was used to partition the data based on Bayesian Information Criterion BIC and find the best partitioning scheme and base substitution models for ML and BI. ML was analyzed using IQ-TREE v1.6.8 (Minh et al., 2013;Nguyen et al., 2015), with the standard bootstrap of 1000 replications; bootstrap values (BS) ≥ 70% were considered to represent moderate confidence. BI was performed under MrBayes v3.2.6, with the following parameters: two independent runs, each with four independent Markov Chain Monte Carlo runs, including three heated chains and one cold chain, were set to run for 1 × 10 7 generations, with simultaneous sampling every 1000 generations. The initial 25% of the sampled trees were discarded as burn-ins. Chain convergence was assumed when the mean standard deviation of the split frequencies fell below 0.01. Bayesian posterior probability, in which the support of each node of the BI tree was greater than or equal to 0.95, was considered high confidence. The phylogenetic tree was constructed using Figtree v.1.4.4.

| Basic structure
The mitochondrial genomes of P. octoguttalis and P. adusta both include 37 genes and one control region ( Figure 1). Four PCGs (nad1, nad4, nad5, and nad4l), two rRNA genes (rrnL and rrnS), and eight tRNA genes (trnQ, trnC, trnY, trnF, trnH, trnP, trnL1, and trnV) are encoded from the minority strands. The remaining 23 genes are encoded from the majority of the strands ( Table 2) The mitogenome sequences of both species show obvious AT biases. The nucleotide content of the P. octoguttalis mitogenome is A: 41.0%, T: 40.5%, C: 11.0%, and G: 7.5%, and the P. adusta mitogenome is A: 40.8%, T: 40.7%, C: 11.0%, and G: 7.4%. The AT content is 81.5% and 81.6%, respectively, which is much higher than the GC content. The AT skew is 0.006 and 0.001, and the GC skew is −0.189 and −0.196, respectively, showing a slight A skew and a significant C skew (Table 3). However, the mitogenomes of other sequenced Spilomelinae are biased toward T and C, showing negative AT skew and negative GC skew.

| PCGs and codon usage
Thirteen PCGs are identified in the mitogenomes of P. octoguttalis and P. adusta. Among them,atp8,atp6,cox1,cox2,cox3,nad2,nad3,nad6, and cytb are encoded by the majority strand, and the remaining four genes are encoded by the minority strand. In P. octoguttalis, there are two overlaps, a 7 bp overlap between atp8 and atp6 and 1 bp overlap between atp6 and cox3. In P. adusta, there is only a 7 bp overlap between atp8 and atp6. As with most other Spilomelinae, the start codons of all genes are typical ATN, except for cox1, whose start codon is CGA. The stop codons of cox1, cox2, and nad5 are terminated by an incomplete stop codon T, and the remaining genes are terminated by TAA, which is the most frequent stop codon, although termination coding by TAG has been reported in a few sequenced Spilomelinae. Among the PCGs, the AT content is 80.3% and 79.6%, respectively. The AT bias of these two species is more significant in the third codon, and the AT content of the third codon (83.2% and 85.8%) is higher than that of the first (73.1% and 82.7%) and second codons (74.9% and 79.8%). The AT skew of these two species is 0.01 and 0.003, and their GC skew is −0.173 and −0.181, respectively, showing a slight A skew and an obvious C skew. The concatenated lengths of the 13 PCGs of P. octoguttalis

| rRNA genes and tRNA genes
In the mitogenomes of P. octoguttalis and P. adusta, two rRNA genes are encoded by the minority strand, with concatenated lengths of 2092 and 2077 bp, respectively. The rrnL gene is located between the trnL1 and trnV genes, which are 1355 and 1341 bp long, respectively; the rrnS gene is located between the trnV gene and the A + Tenriched regions, which are 737 and 736 bp long, respectively.
In the mitogenomes of these two species, there are 22 tRNA genes with concatenated lengths of 1468 and 1481 bp, respectively. A total of 14 genes (trnM, trnI, trnW, trnL2, trnK, trnD, trnG, trnA, trnR, trnN, trnS1, trnE, trnT, and trnS2)  The AT content of the RNA gene of these two species is more than 80%, showing an obvious AT bias. As for base skew, both species show a slight A skew and an obvious C skew.
The phylogenetic topology varies among the subfamilies within the "non-PS clade" in different datasets, probably due to only one sample each in Schoenobiinae, Scopariinae, and Glaphyriinae, thus causing a long branch attraction.
On the basis of the above analyses, our analyses confirmed the sister relationship of Pyraustinae and Spilomelinae with strong support. Trichaeini in this study robustly constitute a monophyletic group in Spilomelinae, with the relationships (Trichaeini + Nomophi lini) + ((Spilomelini + (Hymeniini + Agroterini)) + Margaroniini). Within the "non-PS clade," the monophyly of Acentropinae, Crambinae, and Odontiinae was well supported. The close relationship between Odontiinae and Glaphyriinae, between Schoenobiinae and Acentropinae, and between Scopariinae and Crambinae seemed to be more realistic.

| CON CLUS IONS
In this study, we reported the complete mitogenomes of two Trichaeini and showed satisfactorily high support values. However, its sister group was not completely resolved, combined with previous multisite studies. In addition, the phylogenetic relationships within Crambidae in phylogenetic tree in our present study were in general agreement with previous studies, whereas the affinities in the "non-PS clade" were still unstable and require further investigation. Therefore, improving sample coverage and combining different molecular markers, such as mitochondrial genome and nuclear genes, should be considered in the future research on these taxa.

ACK N OWLED G M ENTS
We would like to thank Miss Yao Sheng and Miss Ruonan Xu

CO N FLI C T O F I NTER E S T S TATEM ENT
All authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The complete mitogenomes of two Prophantis species in this study are deposited in GenBank of NCBI under accession number OP559507 (P. octoguttalis) and OP559508 (P. adusta).